Electrophoresis gels and cross-linking agents for their preparation

ABSTRACT

This invention relates to the separation of molecules on polymer gels, in particular to novel cross-linked polymer gels comprising the cross-linking moiety of for formula (1)                    
     and their preparation, the separation of molecules by techniques such as electrophoresis using these gels, novel cross-linking agents useful in the preparation of the gels, and novel intermediates useful in the synthesis of the cross-linking agents. The invention is especially suitable for electrophoretic applications.

TECHNICAL FIELD

This invention relates to the separation of molecules on polymer gels,in particular to the preparation of novel crosslinked polymer gels, theseparation of molecules by techniques such as electrophoresis usingthese gels, novel crosslinking agents useful in the preparation of thegels and novel intermediates useful in the synthesis of the crosslinkingagents. The invention is especially suitable for electrophoreticapplications and accordingly, for convenience, the invention will befurther described with reference to electrophoresis. It is to beunderstood, however, that the gels, processes and crosslinking agents ofthe present invention are not so limited.

BACKGROUND OF INVENTION

Polyacrylamide gel electrophoresis is an analytical technique wherebyfragments of biomolecules, such as DNA, enzymes and proteins, may beseparated and identified on the basis of their molecular size, weightand charge. Commercially available electrophoresis gels haveconventionally been produced by copolymerisation of acrylamide with thesymmetrical crosslinking agent, N,N′-methylene bisacrylamide, otherwiseknown as BIS. Since both double bonds of BIS are of the same type theirreactivities are essentially the same. Other known crosslinking agentsinclude ethylene glycol diacrylate, dihydroxy ethylene-bisacrylamide(DHEBA), N,N′-propylenebisacrylamide, diacrylamide dimethylether,1,2-diacrylamide ethyleneglycol, ethyleneureabisacrylamide,N,N′-bisacrylylcystamine and bisacrylamide methylether (BAME). As forBIS, the double bonds of these crosslinking agents are of the same type.

DISCLOSURE OF INVENTION

It has now been found that electrophoresis gels having surprisinglyimproved separating ability can be prepared using particularasymmetrical crosslinking agents.

Accordingly the invention provides a crosslinked polymer gel comprisinga crosslinking moiety of the formula:

wherein X and X′ are independently selected from the group consisting of—O—, —S— and —NR—, where R is H, alkyl or cycloalkyl,

R₂ is a C₁-C₄ alkyl group,

Y is an optionally substituted non-aromatic divalent linking group, and

Z is O or S.

Preferably R₂ is CH₃.

The monomer or monomers used to prepare the gel may be any suitablemonomer.

The crosslinked polymer gel may be prepared from monomers having theformula H₂C═CR₅—CO—NR₃R₄ where R₃, R₄ and R₅ are each independently H oralkyl optionally monosubstituted by, for example, OH or C(O)CH₂C(O) CH₃.Examples of monomers include acrylamide, acrylamide derivatives oracrylamide substitutes known to the art such as N,N-dimethylacrylamide,methacrylamide, methyloylacrylamide, propylacrylamide, dipropylacrylamide, isopropyl acrylamide, diisopropyl acrylamide, lactylacrylamide, methoxyacrylamide and mixtures thereof. Preferably themonomer is acrylamide.

The linking group may be any suitable non-aromatic hydrocarbyl group,optionally including one or more heteroatoms selected from O, S, N andP.

Preferably X and X′ are the same. Preferably Z is oxygen.

In another aspect of the invention there is provided a method ofpreparing a crosslinked polymer gel, said method including the step ofsubjecting one or more monomers to crosslinking polymerisation with oneor more crosslinking agents of the formula I:

wherein X and X′ are independently selected from the group consisting of—O—, —S— and —NR—, where R is H, alkyl or cycloalkyl,

R₂ is a C₁-C₄ alkyl group,

Y is an optionally substituted non aromatic divalent linking group, and

Z is O or S.

R₂ is preferably CH₃.

The polymer gels according to the present invention may be preparedusing one or more crosslinking agents of formula I, optionally in thepresence of one or more conventional crosslinking agents known to theart. Preferably the crosslinking agent(s) is/are selected to provide agel which is substantially transparent to visible light. Preferably thegel is an aqueous gel.

The polymer gels according to the present invention are useful forseparating molecules, especially charged species or species capable ofbearing a charge such as biomolecules.

The polymer gel may be an electrophoretic gel. The electrophoretic gelmay have a porosity gradient suitable for gradient gel electrophoresis.See for example, Polyacrylamide Gel Electrophoresis across a MolecularSieve Gradient Margolis, J., Kenrick, K. G., Nature, 214, 1967,p1334-1336; Polyacrylamide Gel Electrophoresis in a Continuous MolecularSieve Gradient, Margolis, J., Kenrick, K. G., Analytical biochemistry,25, 1968, p347-362; and Practical System for Polyacrylamide Gradient Gelelectrophoresis, Margolis, J., Laboratory Practice, 22, p107-109, 1973,the disclosures of which are incorporated herein by reference.

In a further aspect the invention provides a method of separatingmolecules comprising:

providing a crosslinked polymer gel by combining one or more monomerswith a crosslinking agent of the formula I:

wherein X and X′ are independently selected from the group consisting of—O—, —S— and —NR—, where R is H, alkyl or cycloalkyl,

R₂ is a C₁-C₄ alkyl group, and

Y is an optionally substituted non aromatic divalent linking group, and

Z is O or S, optionally in the presence of an initiator, subjecting themonomer solution to polymerisation and crosslinking, placing a samplecontaining the molecules to be separated onto the gel, and subjectingthe gel and sample to a separation technique. Preferably the separationtechnique is electrophoresis. The electrophoresis technique employed maybe any of those known to the art, including one-, two- andmulti-dimensional techniques. The electrophoresis technique may begradient gel electrophoresis.

Preferably polymerisation is carried out on a solution of the monomer ormonomers with the crosslinking agent,

The linking group is preferably selected to provide a crosslinking agentwhich is soluble in the monomer solution. For most applicationsinvolving acrylamide the solvent will be water, and accordingly it ispreferred that the linking group is selected to provide a crosslinkingagent which is soluble in water or water/acrylamide. Other solventsinclude DMF, THF, alcohols and other water miscible systems.

Where the solvent is water and/or the monomer is acrylamide, thehydrophilic/lipophilic balance of the linking group may be controlled sothat the cross-linking agent is soluble in water or water/acrylamide.Accordingly if the linking group contains a large number of carbon atoms(eg. more than about 7) the effect on solubility can be offset byincluding sufficient oxygen atoms or other polar groups to provide acrosslinking agent which is soluble in the acrylamide/water solution.

Examples of divalent linking groups include alkylene, oxyalkylene.polyoxyalkylene, cycloalkylene, alkanedioyl, alkylenedisulphonyl,alkylenecarbonyl, thioalkylene, ureylene, oxalyl, aminoalkylene,alkylenedisulphonyl, heterocyclyl and groups of the formula—(R¹)_(m)—R²—(R³)_(n)—, where R¹ and R³ are selected from alkylene,cycloalkylene, heterocyclyl, oxyalkylene, polyoxyalkylene,alkylenecycloalkylene and alkyleneheterocyclyl; R² is selected from adirect bond, —O—, —S—, —S—S—, alkylene, alkanedioyl, alkylenedioxy,alkylenedisulphonyl, —NR—, —NRC(O)O—, —NR—C(O)—NR—, —NRC(O)—, —N═N—,—NRC(O)C(O)—NR—, —C(O)—, —C(S)— and —RNNR—, where R is H, alkyl orcycloalkyl; m and n are 0 or 1 provided that m+n≠0.

As used herein the term “non-aromatic hydrocarbyl group” means anydivalent group comprising carbon and hydrogen which does not include anaromatic or heteroaromatic ring.

As used herein the term “alkylene”, used either alone or in compoundwords such as “oxyalkylene”, “carbonylalkylene” denotes straight chainand branched C₁₋₁₀ alkylene groups. Examples include methylene,ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene,pentylene, isopentylene, sec-pentylene, 1,2-dimethylpropylene,1,1-dimethylpropylene, hexylene, 4-methylpentylene, 1-methylpentylene,3-methylpentylene, 1,1-dimethylbutylene, 2,2-dimethylbutylene,3,3-dimethylbutylene, 1,2-dimethylbutylene, 1,3-dimethylbutylene,1,2,2-trimethylpropylene, 1,1,2-trimethylpropylene, heptylene,5-methylhexylene, 1-methylhexylene, 2,2-dimethylpentylene,3,3-dimethylpentylene, 4,4-dimethylpentylene, 1,2-dimethylpentylene,1,3-dimethylpentylene, 1,4-dimethylpentylene, 1,2,3-trimethylbutyl,1,1,2-trimethylbutylene and the like.

The term “cycloalkylene”, used alone or in compound words such as“alkylenecycloalkylene” denotes divalent cyclic C₃₋₇ alkyl groups.Examples include cyclopropyl, cyclobutyl, cyclopentyl and cycloheptyl.

The term “heterocyclyl” as used alone or in compound names such as“alkyleneheterocyclyl” denotes 5 or 6 membered heterocyclic rings.Examples of 5 or 6 membered heterocyclic rings include pyrrolidine,imidazolidine, pyrazolidine, thiazolidine, isothiazolidine, oxazolidine,piperidine and piperazine.

In this specification the term “optionally substituted” means that agroup may or may not be further substituted with one or more groupsselected from alkyl, cycloalkyl, alkenyl, alkynyl, halo, haloalkyl,haloalkynyl, hydroxy, alkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy,nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl,alkylamino, dialkylamino, alkenylamine, alkynylamino, acyl, alkenacyl,alkynylacyl, acylamino, diacylamino, acyloxy, alkylsulphonyloxy,heterocyclyl, heterocycloxy, heterocyclamino, haloheterocyclyl,alkylsulphenyl, carboalkoxy, alkylthio, acylthio, phosphorous-containinggroups such as phosphono and phosphinyl, and groups of the formula

where X, X′ and Z are as defined above.

The term “alkyl”, used either alone or in compound words such as“haloalkyl” or “alkylthio”, denotes straight chain or branched C₁₋₆alkyl groups. Examples include methyl, ethyl, propyl, isopropyl and thelike.

The term “alkoxy” denotes straight chain or branched alkoxy,. preferablyC₁₋₁₀ alkoxy. Examples include methoxy, ethoxy, n-propoxy, isopropoxyand the different butoxy isomers.

The term “alkenyl” denotes groups formed from straight chain, branchedor mono- or poly-cyclic alkenes including ethylenically mono- orpoly-unsaturated alkyl or cycloalkyl groups as previously defined,preferably C₂₋₁₀ alkenyl. Examples of alkenyl include vinyl, allyl,1-methylvinyl. butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl,cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl,cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl,1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl,1-4,pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl,1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl,1,3,5-cycloheptatrienyl, 1,3,5,7-cyclooctatetraenyl.

The term “halogen” denotes fluorine, chlorine, bromine or iodine,preferably chlorine or fluorine.

The term “acyl” used either alone or in compound words such as“acyloxy”, “acylthio”, “acylamino” or diacylamino” denotes carbamoyl,aliphatic acyl group and acyl group containing a heterocyclic ring whichis referred to as heterocyclic acyl, preferably C₁₋₁₀ acyl. Examples ofacyl include carbamoyl; straight chain or branched alkanoyl, such asformyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl,2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl,decanoyl; alkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl,t-butoxycarbonyl, t-pentyloxycarbonyl or heptyloxycarbonyl;cycloalkylcarbonyl such as cyclopropylcarbonyl cyclobutylcarbonyl,cyclopentylcarbonyl or cyclohexylcarbonyl; alkylsulfonyl, such asmethylsulfonyl or ethylsulfonyl; alkoxysulfonyl, such as methoxysulfonylor ethoxysulfonyl; heterocyclylcarbonyl; heterocyclylalkanoyl, such aspyrrolidinylacetyl, pyrrolidinylpropanoyl, pyrrolidinylbutanoyl,pyrrolidinylpentanoyl, pyrrolidinylhexanoyl or thiazolidinylacetyl;heterocyclylalkenoyl, such as heterocyclylpropenoyl,heterocyclylbutenoyl, heterocyclylpentenoyl or heterocyclylhexenoyl; orheterocyclylglyoxyloyl, such as, thiazolidinylglyoxyloyl orpyrrolidinylglyoxyloyl.

The term “biomolecule” as used herein denotes biological molecules suchas proteins, enzymes and other peptides, genetic material such aschromosomal material, genomic DNA, cDNA, mRNA, tRNA and other oligo- andpolynucleotides. The term includes naturally occurring biologicalmolecules in addition to fragments and recombinant derivatives thereof.

The divalent linking group Y may be saturated or mono-, di- orpoly-unsaturated. Accordingly, the group Y may be any of the linkinggroups described above in which one or more of carbon-to-carbon singlebonds is replaced by a double bond. For example the divalent linkinggroup may further include alkenylene moieties such as butenylene;cycloalkenylene moieties such as 1-cyclohexenylene, alkenedioyl moietiessuch as fumaryl, maleyl, citraconyl and mesaconyl; heterocyclyl moietiessuch as pyrroline, imidazoline, pyrazoline and oxazoline.

Other suitable divalent linking groups include amino substituted groupssuch as glutamyl, aspartyl and asparaginyl, hydroxy substituted groupsderived from glyceric acid, glycerol and pentaerythritol, and groupssubstituted with both hydroxy and amino such as threonyl.

Preferably linking group Y is selected from C₁₋₇ alkylene, alkenylene,—(CH₂CH₂—O)_(p)—, —(CH₂CH₂CH₂—O)_(p)—, 5 or 6 membered cycloalkylene orheterocyclyl, C₁₋₇ alkenedioyl, C₁₋₇ alkanedioyl, C₁₋₇ alkylenedioxy,C₁₋₇ alkylenedicarbonyl and groups of the formula—(R¹)_(m)—R²—(R³)_(n)—, optionally substituted with one or twosubstituents selected from C₁₋₅ alkyl, hydroxy, halo, amino, C₁₋₅alkyloxy and nitro; where R¹ and R³ are selected from C₁₋₅ alkylene, 5or 6 membered cycloalkylene or heterocyclyl, —(CH₂CH₂—O)_(p)—,—(CH₂CH₂CH₂—O)_(p)—, R² is selected from —O—, —S—, —S—S—, —NR—,—NRC(O)O—, —NRC(O)NR—, alkylenedioxyl, and —C(O)—, p is 1 to 8 and m, nand R are as defined above.

More preferably the linking group is selected from C₁₋₅ alkylene, C₁₋₅alkenylene, —(CH₂CH₂—O)_(p)—, C₁₋₅ alkanedioyl, C₁₋₅ alkylenedicarbonyl,—(C₁₋₅ alkylene)_(m)—R²—(C₁₋₅ alkylene)_(n)—, —(CH₂CH₂O)_(m—R)²—(CH₂CH₂O)_(n)—, optionally substituted with one or more substituentsselected from C₁₋₃ alkyl, hydroxy, halo, C₁₋₃ alkyloxy; where R² isselected from —O—, —S—S—, —NR— and NRC(O)NR—, where R is H or CH₃, p is1 to 4 and m and n are as defined above.

Most preferably the linking group Y is selected from —CH₂—, —CH₂—CH₂—,—CH₂—O—CH₂—, —CHOHCHOH—, —CH₂—S—S—CH₂— and —(CH₂)₂NHC(O)NH(CH₂)₂—.

The crosslinking agents according to the present invention may beprepared by conventional methods. In one such method an appropriatesubstrate, i.e. a diamine, dialcohol, dithiol, aminoalcohol,thiolalcohol or thiolamine, is reacted with an equimolar amount of areactive acryloyl or methacryloyl species, such as acryloyl chloride ormethacryloyl chloride, in a suitable solvent such as chloroform ortetrahydrofuran, to form a monoacryloyl (or monomethacryloyl)intermediate. This intermediate may be isolated and purified beforefurther reaction with an equivalent amount of the other reactiveacryloyl or methacryloyl species, or the complete reaction may becarried out in two steps in a single pot. Reaction with the acryloylspecies may be followed by reaction with the methacryloyl species orvice versa.

Where there is little or no reactivity differential between the reactiveends of the substrate, eg. for diamines, dialcohols and dithiols, it maybe desirable to first protect one end of the substrate with anappropriate protecting group such as t-BOC. The other end can then bereacted with the reactive acryloyl or methacryloyl species to form amonoacryloyl (or monomethacryloyl) intermediate. Removal of theprotecting group is then followed by reaction with the other reactivemethacryloyl or acryloyl species. It is also possible to achievereaction predominantly at one end of such a substrate by controlling thepH of the reaction mixture.

Some of the monoacryloyl and monomethacryloyl intermediates are novelcompounds and represent a further aspect of the invention.

Preferred crosslinking agents useful in the present invention includethe following:

While not wishing to be limited by theory it is believed that the morereactive vinyl group will be preferentially incorporated into thepolymer chain, for example the methacryloyl end of the crosslinkingagent reacts first with the monomer to yield a polymer with pendantacryloyl units. In this way long linear chains of the monomerincorporating primarily methacryloyl groups of the crosslinking agentwould be produced first, before crosslinking of the linear polymericchains starts to occur. It is believed that this delayed crosslinkingproduces a gel having a microporous structure more suitable for theseparation of biomolecules than known gels prepared from symmetricalcrosslinking agents such as BIS.

Some of the bisamide, bithioester and amidethioester crosslinking agentsaccording to the present invention are novel and accordingly in anotheraspect the invention there is provided a compound of formula:

wherein X and X′ are selected from —S— and —NR—, where R is H, alkyl orcycloalkyl, and

Y and Z are as defined above, provided that when X and X′ are both —NR—,Y is not —CH₂— and does not include a quarternary ammonium group.

Some of the amide-ester and thioester ester crosslinking agents of thepresent invention are also novel and. accordingly, in another aspect ofthe invention there is provided a compound of formula:

where one of X and X′ is —O— and the other is selected from —S— and—NR—, where R is H, alkyl or cycloalkyl, and

Y and Z are as defined above; provided that Y is not C₁₋₅ alkylene anddoes not include a quarternary ammonium group, and that when the otherof X and X′ is —NH—, Y is not methyleneoxy-2-hydroxypropylene.

In addition to being useful crosslinking agents in the preparation ofcrosslinked polymer gel, the novel compounds, in view of their abilityto form cross linked polymer networks, may be used as precursors fornovel polymeric materials, or they may be used in admixtures with otherpolymerisable entities to produce novel crosslinked polymeric compoundsto form products such as optical lenses, dental cements, surfacecoatings, plastic films, heat resistant plastics and adhesives. Thesecompounds also have potential as biologically active compounds (e.g.antitumour agents).

The gels of the present invention may be prepared by conventionalmethods. They may be prepared in a variety of polymer concentrations,depending on the sizes of the molecules to be separated. Polyacrylamidegels crosslinked with BIS are commonly used to separate DNA fragmentsless than 1 kb in length. For this purpose the gels are prepared havingacrylamide concentrations in the range of about 3.5 to 20%. Gels havinga concentration of 3.5% are useful for separating DNA fragments of about100 to 1000 nucleotides while gels having an acrylamide concentration ofabout 20% are useful for separating fragments having from about 10 to100 nucleotides.

Since the microporous structure of the gels according to the presentinvention is dependent on the particular crosslinking agent used, aswell as the monomer concentration, the optimum monomer concentration fora particular biomolecule size range may differ somewhat from the optimummonomer concentrations known for acrylamide/BIS systems. The optimumconcentration can be readily determined from standard trial runs.

Generally, however, the crosslinking agent can be employed in an amountof approximately 1 to 30 wt. %, preferably 2 to 10 wt. %, based on thetotal weight of the monomer and the crosslinking agent (%C). For thetotal gel concentration, monomers may be employed in approximately 1 to50 wt %, preferably 1.5 to 20 wt %, based on total solution volume (%T).

The crosslinking polymerization reaction by which the novel gels of thisinvention are prepared is generally carried out in an aqueous medium andcan be initiated by known initiators or polymerization catalysts.Suitable free radical-providing initiator systems are benzoyl peroxide,t-butylhydroperoxide, lauroyl peroxide, cumene hydroperoxide, tetralinperoxide, acetyl peroxide, caproyl peroxide, t-butylperbenzoate,t-butyldiperphthalate, methylethylketone peroxide, hydrogenperoxide-Fe²⁺-ascorbic acid, riboflavin-light, methylene blue-light, andvarious persulfate salts in conjunction withN,N,N′,N′-tetramethylethylenediamine (TEMED), diethylmethylaminediamine(DEMED), 3-dimethylaminopropionitrile (DMAPN) or similar reagents andammonium persulfate-metabisulfite. Another class of free radicalgenerating initiators are azocompounds such as azodiiosobutyronitrile,azodiisobutyramide, azobis (dimethylvaleronitrile), azobis(methylbutyronitrile), dimethyl, diethyl, ordibutylazobismethylvalerate. These and similar reagents contain a N,Ndouble bond attached to aliphatic carbon atoms, at least one of which istertiary. The amount and type of initiator is generally indicated by thenature and concentrations of the monomer and crosslinking agent used.The optimum amount of initiator is also affected by the presence of anyaccompanying impurities. Generally speaking, however, the initiator canbe employed in the amount of approximately 0.3 to 5 wt. % based on thetotal amount of the monomer and crosslinking agent. The preferredinitiator system is TEMED, DEMED or DMAPN and a persulfate salt.

Methods known in the art for utilizing polyacrylamide gels fordetermination of nucleotide sequences usually involve the preparation ofthe gels in given thicknesses, such as between glass plates, or platesof synthetic transparent material, to a thickness of approximately 3 mm.The gel may also be polymerized onto a support film. DNA sampleslabelled such as with ³²P, ³⁵S or fluorescent dyes are placed ontosample slots and electrophoresed. After electrophoresis (which generallyoccurs over a period of from 1 hour to a number of days) the gel isremoved from the plates and autoradiography performed. In automatedsystems, fluorescent labelled nucleotides are monitored during theseparation. Autoradiography requires 10 to 20 hours after which timefilms are studied to determine nucleotide sequence. The preparation ofgels for autoradiography of ³⁵S nucleotides requires immersion in 10%acetic acid to remove urea and handling of the gels with caution due toextreme fragility.

When proteins are to be separated by electrophoretic methods based ontheir size, an ionic detergent, such as sodium dodecyl sulfate (SDS) isgenerally added to the polyacrylamide gel, optionally in conjunctionwith other denaturants, to unfold the protein and provide a net negativecharge. It is then possible to estimate molecular sizes from mobilitiescompared to known standards. In native electrophoresis where separationis made according to change and/or molecular weight, the polyacrylamidegels are generally used in combination with acidic, basic or neutralbuffer systems in the absence of denaturing agents. Electrodes arepositioned according to the predicted net charge of the sample at the pHused.

The gels according to the present invention may include conventionaladditives known to the art as required by the technique employed. Theseadditives include detergents, such as SDS; denaturing agents, such asurea, N,N′-dimethylformamide, n-propylalcohol, formamide, dimethylformamide and glycine; high molecular weight polymers, such as polyvinylalcohol, linear polyacrylamide, polyethylene glycols; and low molecularweight species such as glycerol and sucrose. The gels may also include asuitable buffer system.

A number of suitable buffer systems are disclosed in WO91/14489 thedisclosure of which is incorporated herein by reference. These are shownbelow in Table I.

TABLE 1 Buffer pH Citrate-phosphate 3.2 Succinate 5.2Phosphate-magnesium sulfate 6.8 Tris-EDTA-acetate 7.2 Tris-HCl-magnesiumsulfate 7.4 Tris-EDTA-acetate 7.8 Tris-magnesium chloride 8.0Tris-EDTA-borate 8.3 Tris-EDTA-borate 8.6 Tris-EDTA-lactate 8.6Tris-veronal 8.6 Veronal 9.2 Tris-EDTA-borate 9.5 Tris-EDTA-phosphate8.6 Tris-glycine 8.8 Tris-glycine-SDS 8.8 Sodium phosphate 7.5Sodium-phosphate SDS 7.5 Ethanolamine/GABA* 9.5-10   Tris/acetate/GABA9.6-10.2 Ammediol/GABA 9.6-10.2 Ammediol/HCl 9.6-10.2 Tris-HCl 9.3-9.6 *GABA = gamma, amino butyric acid

In addition to the analytical and preparative separation ofbiomolecules, the gels according to the present invention may be used toestimate molecular weights, and elucidate composition of complexmixtures. The gels may also be used in the sequencing of proteins andDNA, as polyelectrolytes, and may find use in environmental and qualitycontrol applications.

The performance of known gels has so far been limited by a number offactors including (a) restriction on pore size range available (therebyrestricting the molecular weight range of fragments which can beseparated), (b) the susceptibility of some gels to hydrolysis inslightly alkaline media, or to premature mechanical degradation and (c)high background staining caused by the amide linkages of BIS. Anadvantage of the present invention is the ability to control or modifygel formation to produce a gel having the desired characteristics (e.g.pore size, mechanical stability, alkaline stability, background stainingetc.) by selecting an appropriate asymmetrical crosslinking agent.

In order to more clearly describe the invention reference will be madeto the following examples and drawings which describe some preferredembodiments of the invention. However the particularity of the examplesand drawings is not to be understood to supersede the generality of thepreceding description.

BRIEF DESCRIPTION OF DRAWINGS

Referring to the drawings:

FIG. 1 shows the separation of a standard protein mixture achieved usinggels according to the present invention (Gels B, C and D) compared to astandard BIS gel (Gel A), where the concentrations of acrylamide andcrosslinking agent are equivalent, on a mole:mole basis, with a 10% T 3%C BIS gel.

FIG. 2 shows the separation of the same standard protein mixture of FIG.1 where the concentrations of acrylamide and crosslinking agent areequivalent, on a mole:mole basis, with a 15% T 3% C BIS gel.

EXAMPLES Example 1 Preparation of 2-Methacrylethylacrylamide

Acryloyl chloride (25 mmol, 2.0 cm³) in chloroform (25 cm³) was addeddropwise to a stirred solution of ethanolamine (50 mmol, 3.0 cm³) inchloroform (50 cm³) at 0° C. After the addition was complete, thereaction mixture was stirred at 0° C. for a further 2 h. Themonohydrochloride precipitate was removed by filtration and the filtrateevaporated under reduced pressure to give the crude product as a yellowoil. This was then taken up in a slurry of basic alumina in chloroform,and stirred at room temperature for 18 h. Removal of the alumina andconcentration of the solution then gave the desired(2-hydroxy)ethylacrylamide as a clear colorless oil. (2.5 g, 87%). δH(400 MHz; CDCl₃) 7.06 (1H, br s), NH; 6.24 (1H, dd, J 17.0, 1.8 Hz),HC═CH _(a)H_(b); 6.17 (1H, dd, J 17.0, 9.8 Hz), HC═CH_(a)H_(b); 5.62(1H, dd, J 9.9, 1.8 Hz), HC═CH_(a) H _(b); 4.13 (1H, s), OH; 3.69 (2H,t, J 5.1 Hz), CH ₂OH; 3.43 (2H, td, J 5.6, 4.9 Hz), NHCH ₂. δ_(C) (75.4MHz; CDCl₃) 166.76, C═O; 130.50, HC═CH₂; 126.81, HC═CH₂; 61.70, CH₂OH;42.41, NHCH₂. Found: M^(+.) 115.14.

To a stirred solution of N-(2-hydroxyethyl)acrylamide (7.0 mmol. 0.8 g)and triethylamine (7.7 mmol, 1.1 cm³) in chloroform (30 cm³) at roomtemperature, was added dropwise, a solution of methacryloyl chloride(7.7 mmol, 0.8 cm³) in chloroform (15 cm³) under an atmosphere ofnitrogen. After the addition was complete, the reaction mixture wasstirred for 16 h. The reaction mixture was then washed with 2M HCl, andthe organic fractions collected, dried and concentrated under reducedpressure to give the crude product as a yellow oil. This was then takenup in a slurry of basic alumina in chloroform and stirred at roomtemperature for 18 hr. Removal of the alumina and concentration of thefiltrate gave a yellow oil which was subjected to flash chromatography(silica gel, diethyl ether: hexane: methanol elution) to afford2-methacrylethylacrylamide (R_(f) 0.51) as a clear yellow oil (0.77 g,61%). δ_(H) (400 MHz; CDCl₃) 6.20 (1H, br m), NH; 6.26 (1H, dd, J 17.0,1.5 Hz), RHNOCCH═CH _(c)H_(d); 6.11 (1H, dd, J 17.0, 10.3 Hz),RHNOCCH═CH_(c)H_(d); 6.10 (1H, br s), ROO(H₃C)C═CH _(a)H_(b); 5.63 (1H,dd, J 10.3, 1.5 Hz), RHNOCCH═CH_(a) H _(b); 5.58 (1H, m),ROO(H₃C)C═CH_(a) H _(b); 4.26 (2H, t, J 5.4 Hz), CH ₂OC═O; 3.63 (2H, td,J 5.6, 5.4 Hz), CH ₂NHC═O; 1.92 (3H, s) CH ₃. δ_(C) (75.4 MHZ; CDCl₃)167.48, OC═O; 165.69, HNC═O; 135.86, ROO(H₃C)C═CH₂; 130.56, RHNOCCH═CH₂;126.67, RHNOCCH═HH₂; 126.13, ROO(H3C)C═CH₂; 63.32, CH₂OC═O; 38.89,CH₂NHC═O; 18.23, CH₃. Found: M^(+.) 183.22.

Example 2 Preparation of 2-Acrylethylmethacrylamide

Methacryloyl chloride (25 mmol, 2.4 cm³) in chloroform (25 cm³) wasadded dropwise to a stirred solution of ethanolamine (50 mmol, 3.0 cm³)in chloroform (50 cm³) at 0/C. After the addition was complete, thereaction mixture was stirred at 0/C. for a further 2 h. Themonohydrochloride precipitate was removed by filtration and the filtrateevaporated under reduced pressure to give the crude product as a yellowoil. This was then taken up in a slurry of basic alumina in chloroform,and stirred at room temperature for 18 h. Removal of the alumina andconcentration of the solution then gave the(2-hydroxy)-ethylmethacrylamide product as a clear colorless oil. (3.2g, 96%). δ₁₁ (400 MHz; CDCl₃) 6.57 (1H, br m), NH; 6.27 (1H, dd, J 17.0,1.5 Hz), HC═CH _(a)H_(b); 6.14 (1H, dd, J 17.0, 10.0 Hz),HC═CH_(a)H_(b); 5.65 (1H, dd, J 10.0, 1.5 Hz), HC═CH_(a) H _(b); 3.74(2H, t, J 4.9 Hz), CH ₂OH; 3.48 (2H, td, J 5.6, 4.9 Hz), NHCH ₂; 3.37(1H, s), OH. δ_(C) (75.4 MHZ; CDCl₃) 166.76, C═O; 130.50, HC═CH₂;126.81, HC═CH₂; 61.70, CH₂OH; 42.41, NHCH₂. Found: M^(+.) 129.17.

To a stirred solution of N-(2-hydroxyethyl)methacrylamide (10 mmol, 1.3g) and triethylamine (11 mmol, 1.5 cm³) in chloroform (40 cm³), wasadded dropwise, a solution of acryloyl chloride (11 mmol, 0.9 cm³) inchloroform (20 cm³) at room temperature under a nitrogen atmosphere.After the addition was complete, the reaction mixture was stirred for afurther 16 h. The usual work-up as for example 1 then gave thecrosslinking monomer, 2-acrylethylmethacrylamide (R_(f) 0.54) as aclear, colorless oil (0.6 g, 32%). δ_(H) (400 MHz; CDCl₃) 6.45 (1H, brm), NH; 6.38 (1H, dd, J 17.8, 2.0 Hz), ROOCH═CH _(a)H_(b); 6.08 (1H, dd,J 17.8, 8.9 Hz), ROOCH═CH_(a)H_(b); 5.82 (1H, dd, J 8.9, 2.0 Hz),ROOCH═CH_(a) H _(b); 5.66 (1H, s), RHNOC(H₃C)C═CH _(c)H_(d); 5.29 (1H,s), RHNOC(H₃C)C═CH_(c) H _(d); 4.25 (2H, t, J 4.9 Hz), CH ₂OCO; 3.57(2H, td, J 4.9, 4.7 Hz), CH ₂NHCO; 1.94 (3H, s), CH ₃. δ_(C) (75.4 MHZ;CDCl₃) 168.52, HNC═O; 166.22, OC═O; 139.60, RHNOC(H₃C)C═CH₂; 131.34,ROOCH═CH₂; 127.83, ROOCH═CH₂; 119.69, RHNOC(H₃C)C═CH₂; 63.07, CH₂OC═O;38.97, CH₂NHC═O; 18.43, CH₃. Found: M^(+.) 183.22.

Example 3 Preparation of N-Methyl, N-acryloyl-2-ethylmethacrylate

Acryloyl chloride (25 mmol, 2.0 cm³) in chloroform (25 cm³) was addeddropwise to a stirred solution of 2-(methylamino)ethanol (50 mmol, 4.0cm³) in chloroform (50 cm³) at 0° C. After the addition was complete,the reaction mixture was stirred at 0° C. for a further 2 h. Themonohydrochloride precipitate was removed by filtration and the filtrateevaporated under reduced pressure to give the crude product as a yellowoil. This was then taken up in a slurry of basic alumina in chloroform,and stirred at room temperature for 18 h. Removal of the alumina andconcentration of the solution then gave the desired N-methyl,N-acryloyl-2-ethanol as a clear colorless oil (2.77 g, 86%). δ_(H) (300MHz; CDCl₃) 6.47 (1H, dd, J 17.0, 1.8 Hz), HC═CH _(a)H_(b); 6.23 (1H,dd, J 17.0, 9.9 Hz), HC═CH_(a)H_(b); 5.57 (1H, dd, J 10.0, 1.9 Hz),HC═CH_(a) H _(b); 4.07 (1H, s), OH; 3.77 (2H, t, J 5.1 Hz), CH ₂OH; 3.46(2H, td, J 5.6, 4.9 Hz), NCH ₂, 3.10 (3H, br m), N(CH₃). δ_(C) (75.4MHZ; CDCl₃) 166.85, C═O; 130.53, HC═CH₂; 126.85, HC═CH₂; 60.88, CH₂OH;43.76, NCH₂. Found: M^(+.) 129.17.

To a stirred solution of intermediate (7.0 mmol, 0.8 g) andtriethylamine (7.7 mmol, 1.1 cm³) in chloroform (30 cm³) at roomtemperature, was added dropwise, a solution of methacryloyl chloride(7.7 mmol, 0.8 cm³) in chloroform (15 cm³) under an atmosphere ofnitrogen. The work-up as for example 1 then afforded N-methyl,N-acryloyl-2-ethylmethacrylate as a clear yellow oil (0.65 g, 47%).δ_(H) (400 MHz; CDCl₃) 6.54 (1H, dd, J 17.8, 10.3 Hz),N(CH₃)CH═CH_(a)H_(b); 6.33 (1H, dd, J 17.8, 2.0 Hz), N(CH₃)CH═CH_(a)H_(b); 6.15 (1H, br m), O₂C(H₃C)C═CH _(c)H_(d). 5.90 (1H, br m),O₂C(H₃C)C═CH _(c)H_(d); 5.63(1H, dd, J 10.1, 2.0 Hz), N(CH₃)CH═CH_(a) H_(b); 4.09 (2H, t, J 4.9 Hz), CH ₂OCO; 3.48 (2H, td, J 4.9, 4.7 Hz), CH₂N(CH₃)CO; 3.03 (3H, s), N(CH ₃); 1.88 (3H, s), CH ₃. δ_(C) (75.4 MHz;CDCl₃) 167.50, OC═O; 166.85, N(CH₃)C═O; 136.46, RO₂C(H₃C)C═CH₂; 130.92,RN(CH₃)OCH═CH₂; 126.79, RN(CH₃)OCH═CH₂; 124.10, RO₂C(H₃C)C═CH₂; 61.75,CH₂OC═O; 45.33, CH₂N(CH₃)C═O; 35.61, N(CH₃); 17.20, CH₃. Found: M^(+.)197.25.

Example 4 Preparation of N-Methyl, N-methacryloyl-2-ethylacrylate

Methaclyloyl chloride (25 mmol, 2.4 cm³) in chloroform (25 cm³) wasadded dropwise to a stirred solution of 2-(methylamino)ethanol (50 mmol,4.0 cm³) in chloroform (50 cm³) at 0° C. After the addition wascomplete, the reaction mixture was stirred at 0° C. for a further 2 h.The monohydrochloride precipitate was removed by filtration and thefiltrate evaporated under reduced pressure to give the crude product asa yellow oil. This was then taken up in a slurry of basic alumina inchloroform, and stirred at room temperature for 18 h. Removal of thealumina and concentration of the solution then gave the desiredN-methyl, N-methacryloyl-2-ethanol intermediate as a clear colorless oil(3.22 g, 90%). δ_(H) (300 MHz; CDCl₃) 5.76 (1H, br m), N(H₃C)C═CH_(a)H_(b); 5.45 (1H, br m), N(H₃C)C═CH_(a) H _(b); 4.15 (1H, s), OH;3.82 (2H, t, J 5.1 Hz), CH ₂OH; 3.53 (2H, td, J 5.6, 4.9 Hz), NCH ₂,3.14 (3H, br m), N(CH₃); 1.97 (3H, s), CH ₃. δ_(C) (75.4 MHZ; CDCl₃)171.88, C═O; 141.05, (H₃C)C═CH₂; 126.81, (H₃C)C═CH₂; 62.35, CH₂OH;43.21, NCH₂; 36.56, N(CH₃); 19.53, CH ₃. Found: M^(+.) 143.20.

To a stirred solution of intermediate (7.0 mmol, 0.8 g) andtriethylamine (7.7 mmol, 1.1 cm³) in chloroform (30 cm³) at roomtemperature, was added dropwise, a solution of acryloyl chloride (7.7mmol, 0.6 cm³) in chloroform (15 cm³) under an atmosphere of nitrogen.The work-up as for example 1 then yielded N-methyl,N-acryloyl-2-ethylmethacrylate as a clear yellow oil (0.72 g, 52%).δ_(H) (400 MHz: CDCl₃) 6.38 (1H, dd, J 17.0, 1.5 Hz), O₂CCH═CH_(c)H_(d); 6.11 (1H, dd, J 17.0, 10.3 Hz), O₂CCH═CH_(c)H_(d); 5.77 (1H,dd, J 10.3, 1.5 Hz), O₂CCH═CH_(c) H _(d); 5,72 (1H, br m),N(H₃C)C═CH_(a) H _(b); 5.38 (1H, br m), N(H₃C)C═CH_(a) H _(b); 4.10 (2H,t, J 5.4 Hz), CH ₂OC═O; 3.47 (2H, td, J 5.6, 5.4 Hz), CH ₂N(CH₃)C═O;3.04 (3H, s), N(CH₃); 1.95, (3H, s) CH ₃. δ_(C) (75.4 MHz; CDCl₃)172.80, N(H₃C)C═O; 166.70, OC═O; 140.16, N(H₃C)C═CH₂; 131.85,RO₂CCH═CH₂; 128.80, RO₂CCH═CH₂; 126.67, N(H₃C)CH═CH₂; 1119.56,N(H₃C)C═CH₂; 61.60, CH₂OC═O; 45.47, CH₂N(CH₃)C═O; 36.35, N(CH₃); 19.24,CH₃. Found: M^(+.) 197.25.

Example 5 Preparation of N-Methyl, N-methacryloyl,N′-acryloyl-ethylenediamine

To a stirred solution of N-methyl-ethylenediamine (0.10 mol, 8.8 cm³) indioxane (50 cm³), at room temperature and under a nitrogen atmosphere,was added a solution ofS-tert-butyloxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine (0.05 mol, 12g) in dioxane (50 cm³), dropwise, over 3 h. After the addition wascomplete, the solution was stirred at room temperature for a further 15h. The precipitate which formed (4, 6-dimethyl-2-mercaptopyrimidine) wasremoved from the solution, and the filtrate conc. in vacuo to half itsoriginal volume (50 cm³). To this residue, was added water (80 cm³) andthe solution then left to stand at room temperature for 15 min. Thebis-(t-BOC)-diamine precipitate was then removed, and the filtrate conc.in vacuo. To the residue was added water (80 cm³), and the solutionsubsequently saturated with sodium chloride. This saturated solution wasextracted with ethyl acetate (4×40 cm³), and the organic extractscombined, dried (MgSO₄), filtered, and conc. in vacuo to give an oil.This oil was dissolved in water (50 cm³) and acidified to pH 3 with 1Mhydrochloric acid. The solution was then extracted with ethyl acetate(3×100 cm³). The aqueous layer was then collected, decolourized withactivated charcoal and filtered over Celite. The filtrate was then conc.in vacuo . If necessary, fractional recystallization from roomtemperature methanol was used to separate the required product from anydiamine dihydrochloride by-product. The filtrate was then conc. invacuo. The product was isolated by trituration from hexane, to give afine off-white solid which consisted of a mixture of N-t-BOC,N-methyl-ethylenediamine monohydrochloride and N-t-BOC, N′-methylethylenediamine monohydrochloride in a 4:1 ratio (4.8 g, 45%). m.p.104-105° C. δ_(H) (400 MHz; methanol-d₄) 3.52-3.37 (2H, t, J 4.8 Hz), CH₂NH; 3.31 (2H, t, J 4.8Hz), CH ₂NH(CH₃); 2.81 (3H, s), NCH ₃; 1.43 (9H,s), C(CH ₃)₃. δ_(C) (75.4 MHz; methanol-d₄) 157.88, C═O; 81.60 C(CH₃)₃;47.47, CH₂NH(CH₃); 39.09, CH₂NH; 28.65, C(CH₃)₃. Found: M^(+.) 210.72.

The intermediate N-t-BOC, N-methyl-ethylenediamine monohydrochloride(10.0 mmol, 2.11 g) was stirred with 2M NaOH (11 cm³) at roomtemperature for 10 min. The solution was then cooled to 0° C., and tothis, was added a solution of acryloyl chloride (11.0 mmol, 0.9 cm³) inchloroform (30 cm³), dropwise. After the addition was complete, thereaction mixture was stirred at 0° C. for a further 1 h. The organic andaqueous phases were then separated, and the aqueous phase extracted withchloroform. The organic extracts were combined, dried (MgSO4), filteredand conc. in vacuo, to give the crude product. The crude product wasstirred at room temperature over a slurry of basic alumina in chloroformfor 18 hr. Removal of the alumina and concentration of the filtrate thengave a pale yellow oil. Purification by flash chromatography (silicagel, diethyl ether: hexane: methanol elution) afforded the major isomerN-t-BOC, N-methyl, N′-acryloyl-ethylenediamine (R_(f) 0.27) as a paleyellow oil which solidified upon standing. Trituration from hexane thengave the product as a white solid (1.55 g, 68%). m.p. 56-57/C. δ_(H)(400 MHz; CDCl₃) 6.72 (1H, br s), NH; 6.20 (1H, d, J 17.3 Hz), CH═CH_(a)H_(b); 6.05 (1H, dd, J 17.1, 10.3 Hz), CH═CH_(a)H_(b); 5.58 (1H, d,J 10.0 Hz), CH═CH_(a) H _(b); 3.43 (4H, m), 2×CH ₂; 2.86 (3H, s), N(CH₃); 1.42 (9H, s), C(CH ₃)₃. δ_(C) (100 MHz; CDCl₃, 50/C) 165.90,NHOCCH═CH₂; 156.88, NCO₂C(CH₃)₃; 131.11, CH═CH₂; 125.56, CH═CH₂; 79.87C(CH₃)₃; 47.62, CH₂NHCO₂C(CH₃)₃; 38.57, CH₂N(CH₃)OCCH═CH₂; 34.62 N(CH₃);28.31, C(CH₃)₃. Found: M^(+.) 228.31.

N-t-BOC, N-methyl, N′-acryloyl-ethylenediamine (2.5 mmol, 0.57 g) wasstirred in a solution of 3M HCl in ethyl acetate (2.5 cm³) at roomtemperature for 30 min. The solution was then conc. in vacuo to give theintermediate N-methyl, N′-acryloyl-ethylenediamine monohydrochloride asa clear yellow oil. This intermediate (2.5 mmol) was then stirred atroom temperature in 2M NaOH (3 cm³) for 10 min. This reaction mixturewas cooled to 0° C., and to this, was added a solution of methacryloylchloride (2.8 mmol, 0.27 cm³) in chloroform (7 cm³), dropwise, over 30min. After the addition was complete, the reaction mixture was stirredat 0° C. for a further 1 h. The organic and aqueous phases were thenseparated, and the aqueous phase extracted with chloroform. The organicextracts were combined, dried (MgSO₄), filtered and conc. in vacuo, togive the crude product. The crude product was stirred at roomtemperature over a slurry of basic alumina in chloroform for 18 hr.Removal of the alumina and concentration of the filtrate then gave apale yellow oil. Purification by flash chromatography (silica gel,diethyl ether:hexane:methanol elution) afforded N-methyl,N-methacryloyl, N′-acryloyl-ethylenediamine as a pale yellow oil (0.24g, 48%). δ_(H) (300 MHz; CDCl₃) 6.79 (1H, br s), NH; 6.2 (1H, d, J 17.1Hz), HC═CH _(a)H_(b); 6.08 (1H, dd, J 17.1, 10.0), HC═CH_(a)H_(b); 5.59(1H, d, J 10.0 Hz), HC═CH_(a) H _(b); 5.18 (br m), (H₃C)C═CH _(c)H_(d);5.00 (br m), (H₃C)C═CH_(c) H _(d); 3.58 (4H, br m), 2×CH ₂; 3.05, (3H,br m) N(CH ₃); 1.90 (3H, br m), CH ₃. δ_(C) (75.4 MHz; CDCl₃) 170.92,NOC(H₃C)C═CH₂; 162.87, NHOCHC═CH₂; 139.39, (H₃C)C═CH₂; 132.00, HC═CH₂;125.42, HC═CH₂; 119.56, (H₃C)C═CH₂; 44.97, CH₂CH₂; 36.21, CH₂ CH₂;33.98, N(CH₃); 19.24, CH₃. Found: M^(+.) 196.26.

Example 6 Preparation of N-Methyl, N-acryloyl,N′-methacryloyl-ethylenediamine

The intermediate N-t-BOC, N-methyl-ethylenediamine monohydrochlorideprepared in example 5 (10 mmol, 2.1 g) was stirred with 2M NaOH (11 cm³)at room temperature for 10 min. The solution was then cooled to 0° C.,and to this, was added a solution of methacryloyl chloride (11 mmol, 1.1cm³) in chloroform (30 cm³), dropwise. After the addition was complete,the reaction mixture was stirred at 0° C. for a further 1 h. The organicand aqueous phases were then separated, and the aqueous phase extractedwith chloroform. The organic extracts were combined, dried (MgSO₄),filtered and conc. in vacuo, to give the crude product. The crudeproduct was stirred at room temperature over a slurry of basic aluminain chloroform for 18 hr. Removal of the alumina and concentration of thefiltrate then gave a pale yellow oil. Purification by flashchromatography (silica gel, diethyl ether: hexane : methanol elution)afforded N-t-BOC, N-methyl, N′-methacryloyl-ethylenediamine (R_(f)0.51)as a yellow oil (1.63 g, 67%). δ_(H) (400 MHz; CDCl₃) 6.94 (1H, br s),HNOC(H₃C)C═CH_(a)H_(b); 5.74 (1H, br m), (H₃C)C═CH _(a)H_(b); 5.30 (1H,br m), (H₃C)C═CH_(a) H _(b); 3.43 (4H, br m), 2×CH ₂; 2.87 (3H, s), N(CH₃); 1.93 (3H, br m), CH ₃; 1.44 (9H, s), C(CH ₃)₃. δ_(C) (75.4 MHz;CDCl₃) 168.52, NHOC(H₃C)C═CH₂; 157.33, NHCO₂C(CH₃)₃; 139.36, (H₃C)C═CH₂;119.81, (H₃C)C═CH₂; 80.85 C(CH₃)₃; 47.32, CH₂N(CH₃)OC(H₃C)C═CH₂; 39.32,CH₂NHCO₂C(CH₃)₃; 34.68, N(CH₃); 28.33, C(CH₃)₃; 18.47, CH₃. Found:(HM⁺.) 243.17178.

N-t-BOC, N′-methyl, N′-methacryloyl-ethylenediamine (2.5 mmol, 0.61 g)was stirred in a solution of 3M HCl in ethyl acetate (2.5 cm³) at roomtemperature for 30 min. The solution was then conc. in vacuo to give theintermediate N-methyl, N′-methacryloyl-ethylenediamine monohydrochlorideas a clear yellow oil. This intermediate (2.5 mmol) was then stirred atroom temperature in 2M NaOH (3 cm³) for 10 min. The reaction mixture wascooled to 0° C., and to this, was then added a solution of acryloylchloride (2.8 mmol, 0.23 cm³) in chloroform (7 cm³), dropwise, over 30min. After the addition was complete, the reaction mixture was stirredat 0° C. for a further 1 h. The organic and aqueous phases were thenseparated, and the aqueous phase extracted with chloroform. The organicextracts were combined, dried (MgSO₄), filtered and conc. in vacuo, togive the crude product. The crude product was stirred at roomtemperature over a slurry of basic alumina in chloroform for 18 hr.Removal of the alumina and concentration of the filtrate then gave apale yellow oil. Purification by flash chromatography (silica gel,diethyl ether: hexane: methanol elution) then gave the crosslinkerN-methyl, N-acryloyl, N′-methacryloyl-ethylenediamine as a pale yellowoil (0.26 g, 53%). δ_(H) (300 MHz; CDCl₃) 7.01 (1H, br s), NH; 6.50 (1H,dd, J 16.8, 10.4 Hz), HC═CH_(a)H_(b); 6.22 (1H, dd, J 12.4, 2.0), HC═CH_(a)H_(b); 5.66 (br m), (H₃C)C═CH _(c)H_(d); 5.61 (1H, d, J 10.5 Hz),HC═CH_(a) H _(b); 5.23 (br m), (H₃C)C═CH_(c) H _(d); 3.57 (2H, br m), CH₂; 3.43 (2H, br m), CH ₂; 3.00, (3H, br m) N(CH ₃); 1.85 (3H, br m), CH₃. δ_(C) (75.4 MHz; CDCl₃, 50° C.) 166.10, NHOC(H₃C)C═CH₂; 165.04,NOCHC═CH₂; 140.40, (H₃C)C═CH₂; 130.92, HC═CH₂; 126.01, HC═CH₂; 118.50,(H₃C)C═CH₂; 44.83, CH₂CH₂; 35.47, CH₂ CH₂; 34.12, N(CH₃); 18.50, CH₃.Found: M^(+.) 196.26.

Example 7 Preparation of N-Acryloyl, N′methacryloyl, N,N′dimethyl-ethylenediamine

Method (a)

To a stirred solution of N, N′-dimethyl-ethylenediamine (0.10 mol, 12.4cm³) in dioxane (50 cm³), at room temperature and under a nitrogenatmosphere, was added a solution ofS-tert-butyloxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine (0.05 mol, 12g) in dioxane (50 cm³), dropwise, over 3 h. After the addition wascomplete, the solution was stirred at room temperature for a further 15h. The work-up as in example 5 gave the product which was isolated bytrituration from hexane, to give N-t-BOC, N, N′-dimethyl-ethylenediaminemonohydrochloride as a white crystalline solid (4.99 g, 45%). m.p.108-110° C. δ_(H) (400 MHz; methanol-d₄) 3.60 (2H, br m), CH ₂N(CH₃);3.20 (2H, t, J 6.25 Hz), CH ₂NH(CH₃); 2.92 (3H, s), NCH ₃; 2.75 (3H, s),HNCH ₃; 1.47 (9H, s), C(CH ₃)₃. δ_(C) (75.4 MHz; methanol-d₄) 157.83,C═O; 81.52 C(CH₃)₃; 48.43, NH(CH₃); 46.26, CH₂NH(CH₃); 35.32,CH₂NH(CH₃); 33.97, NCH₃; 28.68, C(CH₃)₃. Found: M^(+.) 224.75.

The intermediate N-t-BOC, N, N′-dimethyl-ethylenediaminemonohydrochloride (5.0 mmol, 1.12 g) was stirred with 2M NaOH (11 cm³)at room temperature for 10 min. The solution was then cooled to 0/C.,and to this, was added a solution of acryloyl chloride (5.5 mmol, 0.45cm³) in chloroform (11 cm³), dropwise, over 30 min. After the additionwas complete, the reaction mixture was stirred at 0/C. for a further 1h. The organic and aqueous phases were then separated, and the aqueousphase extracted with chloroform. The organic extracts were combined,dried (MgSO4), filtered and conc. in vacuo, to give the crude product.The crude product was stirred at room temperature over a slurry of basicalumina in chloroform for 18 hr. Removal of the alumina andconcentration of the filtrate then gave a pale yellow oil. Purificationby flash chromatography (silica gel, diethyl ether:hexane:methanolelution) afforded N-t-BOC, N′-acryloyl, N, N′-dimethyl-ethylenediamineas a yellow oil (0.62 g, 51%). δ_(H) (300 MHz; CDCl₃) 6.56 (1H, dd, J16.8, 10.4 Hz), CH═CH_(a)H_(b); 6.35 (1H, d, J 15.0 Hz), CH═CH_(a)H_(b); 5.67 (1H, d, J 10.3 Hz), CH═CH_(a) H _(b); 3.55 (2H, br m),CH ₂; 3.37 (2H, m), CH ₂; 3.07 (3H, m), N(CH ₃); 2.86 (3H, m), N(CH ₃);1.45 (9H, m), C(CH ₃)₃. δ_(C) (75.4 MHz; CDCl₃) 166.26, NOCHC═CH₂;155.57, NCO₂C(CH₃)₃; 127.77, HC═CH₂; 126.89, HC═CH₂; 79.23, C(CH₃)₃;47.77, CH₂N(CH₃)OCHC═CH₂; 46.82, CH₂N(CH₃)CO₂C(CH₃)₃; 35.87,CH₂N(CH₃)OCHC═CH₂; 34.54, CH₂N(CH₃)CO₂C(CH₃)₃; 28.22, C(CH₃)₃. Found:M^(+.) 242.34.

N-t-BOC, N′-acryloyl, N, N′-dimethyl-ethylenediamine (2.0 mmol, 0.46 g)was stirred in a solution of 3M HCl in ethyl acetate (2 cm³) at roomtemperature for 30 min. The solution was then conc. in vacuo to giveN′-acryloyl, N, N′-dimethyl-ethylenediamine monohydrochloride as a clearyellow oil. This intermediate was stirred at room temperature in 2M NaOH(3 cm³) for 10 min. The reaction mixture was then cooled to 0° C., andto this, was then added a solution of methacryloyl chloride (2.2 mmol,0.22 cm³) in chloroform (5 cm³) dropwise, over 30 min. After theaddition was complete, the reaction mixture was stirred at 0° C. for afurther 1 h. The organic and aqueous phases were then separated, and theaqueous phase extracted with chloroform. The organic extracts werecombined, dried (MgSO₄), filtered and conc. in vacuo, to give the crudeproduct. The crude product was stirred at room temperature over a slurryof basic alumina in chloroform for 18 hr. Removal of the alumina andconcentration of the filtrate then gave a pale yellow oil. Purificationby flash chromatography (silica gel, diethyl ether: hexane: methanolelution) afforded N-acryloyl, N′-methacryloyl, N,N′-dimethyl-ethylenediamine as a yellow oil (0.12 g, 28%). δ_(H) (300MHz; CDCl₃) 6.49 (1H, dd, J 16.8, 10.4 Hz), HC═CH_(a)H_(b); 6.26 (1H, d,J 16.5), HC═CH _(a)H_(b); 5.66 (1H, m), HC═CH _(a)H_(b); 5.12 (1H, brm), (H₃C)C═CH _(c)H_(d); 4.92 (1H, br m), (H₃C)C═CH_(c) H _(d); 3.53(4H, br m), 2×CH ₂; 3.05 (3H, m) N(CH ₃); 2.98 (3H, m) N(CH ₃); 1.85(3H, m), CH ₃. δ_(C) (75.4 MHz; CDCl₃) 170.92, NOC(H₃C)C═CH₂; 165.04,NOCHC═CH₂; 139.39, (H₃C)C═CH₂; 130.92, HC═CH₂; 126.01, HC═CH₂; 119.56,(H₃C)C═CH₂; 43.23, CH₂CH₂; 43.09, CH₂ CH₂; 36.21, N(CH₃); 35.47, N(CH₃);19.24, CH₃. Found: M^(+.) 210.29.

Method (b)

The intermediate N-t-BOC, N, N′-dimethyl-ethylenediaminemonohydrochloride (5.0 mmol, 1.12 g) as prepared above was stirred with2M NaOH (11 cm³) at room temperature for 10 min. The solution was thencooled to 0° C., and to this, was added a solution of methacryloylchloride (5.5 mmol, 0.55 cm³)in chloroform (11 cm³), dropwise, over 30min. After the addition was complete, the reaction mixture was stirredat 0° C. for a further 1 h. The organic and aqueous phases were thenseparated, and the aqueous phase extracted with chloroform. The organicextracts were combined, dried (MgSO₄), filtered and conc. in vacuo, togive the crude product. The crude product was stirred at roomtemperature over a slurry of basic alumina in chloroform for 18 hr.Removal of the alumina and concentration of the filtrate then gave apale yellow oil. Purification by flash chromatography (silica gel,diethyl ether: hexane: methanol elution) then afforded N-t-BOC,N′-methacryloyl, N, N′-dimethyl-ethylenediamine as a pale yellow oil(0.82 g, 64%). δ_(H) (300 MHz; CDCl₃) 5.16 (1H, br m), (H₃C)C═CH_(a)H_(b); 5.00 (1H, br m), (H₃C)C═CH_(a) H _(b); 3.47 (4H, br m), 2×CH₂; 3.04 (3H, m), N(CH ₃); 2.87 (3H, m), N(CH ₃); 1.93 (3H, br m), CH ₃;1.43 (9H, s), C(CH ₃)₃. δ_(C) (75.4 MHz; CDCl₃) 172.22, NOC(H₃C)C═CH₂;155.21, NCO₂C(CH₃)₃; 140.42, (H₃C)C═CH₂; 115.21, (H₃C)C═CH₂; 79.01C(CH₃)₃; 47.73, CH₂N(CH₃)OC(H₃C)C═CH₂; 44.99, CH₂N(CH₃)CO₂C(CH₃)₃;36.94, CH₂N(CH₃)OC(H₃C)C═CH₂; 0.46, CH₂N(CH₃)CO₂C(CH₃)₃; 28.15, C(CH₃)₃;20.15, CH₃. Found: M^(+.) 256.37. N-t-BOC, N′-methacryloyl, N,N′-dimethyl-ethylenediamine (2.0 mmol, 0.51 g) was stirred in a solutionof 3M HCl in ethyl acetate (2 cm³) at room temperature for 30 min. Thesolution was then conc. in vacuo to give N′-methacryloyl, N,N′-dimethyl-ethylenediamine monohydrochloride as a clear yellow oil.This intermediate was stirred at room temperature in 2M NaOH for 10 min.The reaction mixture was then cooled to 0° C., and to this, was thenadded a solution of acryloyl chloride (2.2 mol, 0.18 cm³) in chloroform(5 cm³) dropwise, over 30 min. After the addition was complete, thereaction mixture was stirred at 0° C. for a further 1 h. The organic andaqueous phases were then separated, and the aqueous phase extracted withchloroform. The organic extracts were combined, dried (MgSO₄), filteredand conc. in vacuo. to give the crude product. The crude product wasstirred at room temperature over a slurry of basic alumina in chloroformfor 18 hr. Removal of the alumina and concentration of the filtrate thengave a pale yellow oil. Purification by flash chromatography (silicagel, diethyl ether:hexane. methanol elution) afforded N-acryloyl,N′-methacryloyl, N, N′-dimethyl-ethylenediamine as a yellow oil (0.20 g,47%). (Spectroscopic data as above)

Example 8 Preparation of N-Acryloyl, N′-methacryloyl-ethylenediamine

A solution of ethylenediamine (15 mmol, 10.1 cm³) in water (150 cm³) at0° C. was adjusted to pH 8.5 with 3N HCl. To this, was added a solutionof methacryloyl chloride (16.5 mmol, 16.1 cm³) in chloroform (100 cm³),dropwise, over 2 h. After the addition was complete, the reactionmixture was stirred at 0° C. for a further 2 h. The organic and aqueouslayers were then separated, and the aqueous layer extracted withchloroform (3×50 cm³). The aqueous layer was then collected and conc. invacuo (freeze-dryer) to give a white solid. The residue was subjected torepeated fractional recrystallization from room temperature methanol,and the filtrate concentrated to give N-methacryloyl-ethylenediaminemonohydrochloride as a clear, yellow oil (14.32 g, 58%).N-methacryloyl-ethylenediamine monohydrochloride (16 mmol, 20.0 g) wasstirred in 2M NaOH (60 cm³) for 10 min. This solution was subsequentlydiluted to a volume of 160 cm³ with water, and stirred at 0° C. To thismixture was added a solution of acryloyl chloride (17.6 mmol, 14.3 cm³)in chloroform (300 cm³), dropwise, over 3 h. After the addition wascomplete, the reaction mixture was then stirred at 0° C. for a further 2h. The organic and aqueous layers were separated, and the aqueousportion extracted with chloroform (3×100 cm³). The organic extracts werecombined and concentrated to give the crude product as a white solid.Recrystallization from acetonitrile then gave N-acryloyl,N′-methacryloyl-ethylenediamine as colorless needles (10.70 g, 37%),m.p. 140-142/C. δ_(H) (400 MHz; CDCl₃) 6.84 (1H, br s), NH; 6.74 (1H, brm), NH; 6.27 (1H, dd, J 16.9, 1.5 Hz), HC═CH _(a)H_(b); 6.13 (1H, dd, J17.0, 10.2), HC═CH_(a)H_(b); 5.74 (1H, s), (H₃C)C═CH _(c)H_(d); 5.65(1H, dd, J 10.3, 1.6 Hz), HC═CH_(a) H _(b); 5.34 (1H, br m),(H₃C)C═CH_(c) H _(d); 3.49 (4H, m), 2×CH ₂; 1.95 (3H, s), CH ₃. δ_(C)(75.4 MHZ; CDCl₃) 169.4, NHOC(H₃C)C═CH₂; 167.0, NHOCHC═CH₂; 139.2,(H₃C)C═CH₂; 130.7, HC═CH₂; 126.2, HC═CH₂; 120.1, (H₃C)C═CH₂; 40.4,CH₂CH₂; 39.5, CH₂ CH₂; 18.4, CH₃. Found: M^(+.) 182.1059.

Example 9 Preparation of monomer stock solutions and SDS gels.

This example describes a general procedure for preparing stock solutionsof acrylamide with a desired crosslinking agent. To ensure that thedegree of crosslinking is equivalent to the corresponding acrylamide/BISsystems the amount of crosslinking agent is calculated on a mole:molebasis rather than a weight:weight basis.

1. Prepare monomer stock solutions of the following concentrationsdepending on the desired crosslinking agent concentration.

a) 30% T 3% C Dissolve 29.1 g acrylamide and 5.837×10⁻³ moles ofcrosslinking agent (equivalent to 0.9 g of BIS on a mole:mole basis) in50 ml of distilled water and bring to a total volume of 100 ml.

b) 30% T 6% C Dissolve 28.2 g acrylamide and 11.67×10⁻³ moles ofcrosslinking agent (equivalent to 1.80 g of BIS) in 50 ml of distilledwater and bring to a total volume of 100 ml.

2. Filter monomer solution through Whatman No. 1 filter paper. Monomerstock solution may be stored at 40° C. for 2 to 3 months.

3. To the required amounts of water and 1.5M Tris HC1 buffer are addedthe required aliquots of the 30% T monomer stock solution of the desired° C. ratio (see Table 2, this being an example of the concentrationrange employed). Degas monomer solution under vacuum for 10 min at roomtemperature and then 10 min at 10° C.

4. Add 10% SDS solution, and then redox initiator system composed onfreshly prepared 10% ammonium persulfate (10% AP, 200 1 l/ml) and3-dimethylaminopropionitrile (DMAPN, 51 l/ml, except for 5% T 3% Cstacking gel which is 101 l/ml) to monomer solution and swirl gently tomix.

TABLE 2 ITEM 5% T 10% T 15% T water 16.6 ml 11.6 ml 6.6 ml 1.5 M Tris7.5 ml 7.5 ml 7.5 ml buffer (pH 8.8) acrylamide/ 5.0 ml 10.0 ml 15.0 mlmononomer stock 10% SDS 0.300 ml 0.300 ml 0.300 ml 10% ammonium 0.600 ml0.600 ml 0.600 ml persulfate DMAPN 30 μl 15 μl 15 μl TOTAL 30 ml 30 ml30 ml VOLUME

5. Using a syringe, inject resolving gel solution slowly into preparedglass cassettes to a height of 75 mm, which are prepared as follows:

Glass cassettes are thoroughly cleaned with detergent and then allowedto air dry before being swabbed with ethanol or iso-propanol to removeany grease or detergent film, and also to neutralise the surface of theglass. After cleaning, the sides and base of the cassettes are taped upusing 3M Scotch electrical tape and then placed in a 60° C. oven for 1hr.

The polyacrylamide gel is allowed to form (approx. 30 to 45 min) underan atmosphere of nitrogen. This nitrogen atmosphere is used for thepolymerization procedure to ensure the absence of oxygen. therebyeliminating the appearance of “swirls” or “troughs” on polymerization.

6. Slowly apply a 5% T 3% C stacking gel onto the top of the resolvinggel, which is also allowed to polymerize under an atmosphere ofnitrogen.

7. Carefully remove the tape and wash the cassettes under water. Gelsare then ready for electrophoresis. By this method it is possible toprepare vast range and size of well polymerized and opticallytransparent gels by varying the proportions of water and acrylamide/crosslinking agent stock solution.

Example 10 SDS-PAGE

This example describes a general procedure for performing SDS-PAGE withgels prepared in accordance with the invention.

1. Use a white surface when applying samples to the gels.

2. Insert gels into electrophoresis kit (which has been assembledaccording to manufacturers' instructions) and fill both reservoirs inthe kit with SDS-PAGE running buffer or appropriate buffer. Place samplewells into the stacking gel at a depth of 1-2 mm.

3. Blow out any air bubbles appearing in the sample wells from above,using a plastic pipette.

4. Load approx. 10 μl of sample (eg. peptide or DNA marker) into thesample well using a microsyringe. Sample should “sit” nicely inside thewell and be an intense blue colour. If too much sample is added,surrounding solution will go cloudy as the sample overflows from thewell.

5. Electrophoresis is then performed noting that a 200V is maintained,current is between 150-40 mA and the process performed for the requiredtime (60 minutes to days). The experiment is complete when the dye frontreaches the bottom of the plate.

6. Remove sample spacers from the gel after approx. 2 min.

7. When electrophoresis is complete, the gel cassette may be removed andopened, and the gel eased off the cassette under running water.

8. The gel is washed into a vessel containing Coomassie Blue R-250(0.25% coomassie blue, 40% methanol, 10% acetic acid) and stained for atleast 30 min for proteins or ethidium bromide for DNA under known of theart conditions.

9. Gel is then destained (for proteins) by soaking in 40% methanol, 10%acetic acid overnight.

10. Gel is then sealed in to zip-lock bag, labelled and filed eg. inplastic pockets of a folder for reference.

Example 11 Comparison of electrophoretic performance with standardBIS/acrylamide gels

In this example the performance of gels prepared in accordance with thepresent invention is compared to the performance of standardBIS/acrylamide gels. For the purpose of the comparison the asymmetricalcrosslinking agents were substituted for BIS on a mole:mole basis ratherthan on a weight:weight basis to ensure equivalence in the number ofreactive double bonds. The gels were compared by examining the migrationvelocity (R_(f)) of peptide and DNA markers in the respective gels,where R_(f) is defined as:$R_{f} = \frac{{distance}\quad {migrated}\quad {by}\quad {protein}\quad {or}\quad {DNA}}{{distance}\quad {travelled}\quad {by}\quad {dye}\quad {front}}$

a) Preparation of acrylamide/crosslinking agent stock solution

Stock solutions of acrylamide and the crosslinking agents were preparedto provide acrylamide/crosslinking agent stock solutions equivalent (ona mole:mole basis) with a 30% T 3% C acrylamide/BIS solution:

for BIS (Gel A);

29.1 g of acrylarnide and 0.90 g (5.837×10⁻³ moles) of crosslinkingagent;

for N-acryloyl, N′-methacryloyl methylenediamine (Gel B);

29.1 g of acrylamide and 0.98 g (5.837×10⁻³ moles) of crosslinkingagent;

for N-acryloyl, N′-methacryloyl ethylenediamine (Gel C);

29.1 g of acrylamide and 1.06 g (5.837.10⁻³) moles of crosslinkingagent; and

for methacrylethylacrylamide (Gel D);

29.1 g of acrylamide and 1.07 g (5.837×10⁻³ moles of crosslinking agent.

b) Preparation of gels

Gels were prepared from the stock solutions in accordance with Table 3below.

TABLE 3 Major Components 10% T 3% C* 15% T 3% C* Water 11.6 ml 6.6 ml1.5 M Tris Buffer pH 8.8 7.5 ml 7.5 ml Stock Solution 10.0 ml 15.0 ml10% SDS 0.300 ml 0.300 ml 10% ammonium persulfate 0.600 ml 0.600 mlDMAPN 15 μl 15 μl Total Volume 30 ml 30 ml *These weight basedconcentrations of acrylamide and crosslinking agent are only correctwhen BIS is employed as the crosslinking agents. When a crosslinkingagent other than BIS is employed, the amount of crosslinking agent isthe mole:mole equivalent of BIS required to give these concentrations.

c) Assessment of electrophoretic performance

The stained gels shown in FIGS. 1 and 2 were obtained after performingelectrophoresis under the standard conditions with a commerciallyavailable standard molecular weight protein marker system composed of(1) myosin (200,000), (2), β-galactosidase (116,250), (3) phosphorylaseb (97,400), (4) bovine serum albumin (66,200), (5) ovalbumin (45,000),(6) carbonic anhydrase (31,000), (7) soybean trypsin inhibitor(21,5000), (8) lysozyme (14,400) and (9) aprotinin (6,500). The gelscontain the following crosslinking gents: (Gel A)methylene-bisacrylamide (BIS), (Gel B) N-acryloyl. N′-methacryloylmethylenediamine, (Gel C) N-acryloyl, N′-methacryloyl ethylenediamineand (Gel D) 2-methacrylethylacrylamide.

(i) 10% T 3% C gels

The 10% T 3% C gels prepared and stained as described above are shown inFIG. 1. It is clear from a comparison of the stained gels thatequivalent proteins move through the gels incorporating the asymmetricalcrosslinking agents (Gels B, C and D) at a faster rate, ie. have ahigher migration or Rf value, than through the standard BIS gel (Gel A).There also appears to be differences in the degree of separation betweenspecific protein bands such that these new gels may be used in a mannerto achieve a maximum of separation and resolution within a particularmolecular weight region. For example, a 10% T 3% C gel incorporatingN-acryloyl, N′-methacryloyl methylenediamine (Gel B) exhibits animproved separation for the protein bands in the region 116,250 to66,200, which is similarly observed for the Gels C and D. Each of GelsB, C and D showed enhanced performance in the separation of the highmolecular weight components of the marker system, while remainingoptically transparent.

(ii) 15% T 3% gels

The 15% T 3% C gels prepared and stained described above are shown inFIG. 2.

As with the 10% T 3% gels described above, these 15% T 3% C gelsincorporating asymmetrical crosslinking agents (Gels B, C and D)afforded improved electrophoretic separations compared to the BIS gel(Gel A). The improved separation is evident for both the high and lowmolecular weight components.

These results show an enhanced separation and improved resolution, whilestill maintaining optical transparency. For example, the 15% T 3% C gelincorporating the crosslinking agent N-acryloyl, N′-methacryloylmethylenediamine (Gel B) exhibits an enhanced separation in the region66,000 to 21,500. Similar results are observed for the gels containingthe other asymmetrical crosslinking agents (Gels C and D).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The claims defining the invention are as follows:
 1. A crosslinkedpolymer gel comprising a asymmetrical crosslinking moiety of theformula:

wherein X and X′ are independently selected from the group consisting of—O—, —S— and —NR—, where R is H, alkyl or cycloalkyl, Y is an optionallysubstituted non-aromatic divalent linking group, Z is O or S; and R₂ isa C₁-C₄ alkyl group, provided that when said crosslinked polymer gel isa non-aqueous polymer gel and R₂ is CH₃ and Y is —CH₂—CH₂—, X, X′ and Zare not all O.
 2. A crosslinked polymer gel according to claim 1 whereinR₂ is CH₃.
 3. A crosslinked polymer gel according to claim 1, whereinthe crosslinked polymer gel is a polymer or copolymer of acrylamide, anacrylamide derivative and/or an acrylamide substitute and optionally oneor more other comonomers.
 4. A crosslinked polymer gel according toclaim 1 wherein the gel is an aqueous gel comprising the product formedby crosslinking polymerisation of one or more monomers selected fromcompounds of the formula H₂C═CR₅—CO—NR₃R₄ where R₃, R₄ and R₅ are eachindependently selected from H or optionally substituted alkyl, and oneor more asymmetrical crosslinking agents of the formula:

where X and X′ are selected from the group consisting of —O—, —S— and—NR—, where R is H, alkyl or cycloalkyl, Y is an optionally substitutednon aromatic divalent linking group, and Z is O or S.
 5. A gel accordingto claim 1 wherein the divalent linking group is selected from the groupalkylene, oxyalkylene, polyoxyalkylene, cycloalkylene, alkanedioyl,alkylenedisulphonyl, alkylenecarbonyl, thioalkylene, ureylene, oxalyl,aminoalkylene, alkylenedisulphonyl, heterocyclyl and groups of theformula —(R¹)_(m)—R²—(R³)_(n)—, where R¹ and R³ are selected fromalkylene, cycloalkylene, heterocyclyl, oxyalkylene, polyoxyalkylene,alkylenecycloalkylene and alkyleneheterocyclyl; R² is selected from adirect bond, —O—, —S—, —S—S—, alkylene, alkanedioyl, alkylenedioxy,alkylenedisulphonyl, —NR—, —NRC(O)O—, —NR—C(O)—NR—, —NRC(O)—, —N═N—,—NRC(O)C(O)—NR—, —C(O)—, —C(S)— and —RNNR—, where R is H, alkyl orcycloalkyl; m and n are 0 or 1 provided that m+n≠0.
 6. A porouselectrophoretic medium comprising a gel in accordance with claim
 1. 7.An electrophoretic medium according to claim 6 wherein theelectrophoretic medium has a porosity gradient.
 8. A method of preparinga crosslinked polymer gel, the method including the step of subjectingone or more monomers to crosslinking polymerisation with one or moreasymmetrical crosslinking agents of formula (I).

wherein X and X′ are independently selected from the group consisting of—O—, —S— and —NR—, where R is H, alkyl or cycloalkyl, R₂ is a C₁-C₄alkyl group, Y is an optionally substituted non aromatic divalentlinking group, and Z is O or S.
 9. A method according to claim 8 whereinthe polymer gel is formed by asymmetrical crosslinking polymerisation ofone or more crosslinking agents of formula I, optionally in the presenceof one or more conventional crosslinking agents.
 10. A method accordingto claim 8 wherein X and X′ are the same and R₂ is CH₃.
 11. A methodaccording to claim 8 wherein the crosslinked polymer gel is an aqueousgel comprising the product formed by crosslinking polymerisation in thepresence of an aqueous medium of one or more monomers selected from theformula H₂C═CH—CO—NR₃R₄ wherein R₃ and R₄ are each independently H oralkyl optionally mono-substituted with OH or C(O) CH₂ C(O) CH₃,optionally one or more other comonomers and one or more asymmetricalcross-linking agents selected from compounds of formula I.
 12. A polymergel according to claim 1 wherein the polymer gel is formed bycrosslinking polymerization of one or more compounds of formula:

wherein X and X′ are selected from the group consisting of —S— and —NR—,where R is H, alkyl or cycloalkyl, and Y is an optionally substitutednon-aromatic divalent linking group, and Z is O or S, provided that whenX and X′ are both —NR—, Y is not —CH₂— and does not include a quaternaryammonium group.
 13. A crosslinked polymer formed from one or moremonomers and one or more compounds of formula: wherein X and X′ areselected from the group consisting of —S— and —NR—, where R is H, alkylor cycloalkyl, and Y is an optionally substituted non-aromatic divalentlinking group. and Z is O or S, provided that when X and X′ are both—NR—, Y is not —CH₂— and does not include a quaternary ammonium group.14. A crosslinked polymer formed from one or more monomers and one ormore compounds of formula:

wherein one of X and X′ is —O— and the other is selected from —S— and—NR—, where R is H, alkyl or cycloalkyl; and Y is an optionallysubstituted non aromatic divalent linking group, and Z is O or S;provided that Y is not C₁₋₅ alkylene and does not include a quaternaryammonium group, and that when the other of X and X′ is —NH—, Y is notmethyleneoxy-2-hydroxypropylene.
 15. A polymer gel according to claim 1,wherein the polymer gel is formed by cross-linking polymerization of oneor more compounds of formula:

wherein one of X and X′ is —O— and the other is selected from —S— and—NR—, where R is H, alkyl or cycloalkyl; and Y is an optionallysubstituted non aromatic divalent linking group, and Z is O or S;provided that Y is not C₁₋₅ alkylene and does not include a quaternaryammonium group, and that when the other of X and X′ is —NH—, Y is notmethyleneoxy-2-hydroxypropylene.